SUMMARY/ABSTRACT The overall objective of Project 4 is to discover and exploit extrinsic mechanisms of therapy resistance by focusing on the contribution of tumor-associated macrophages (TAMs) and tumor-associated fibroblasts (TAFs) in the tumor microenvironment (TME). Our overarching hypothesis is that TAMs and TAFs cooperate in creating a favorable tumorigenic environment that ultimately leads to the emergence of therapeutic resistance and immune escape in NB. We also postulate that as tumors are treated, the TME is altered in its composition and function to become increasingly favorable to therapeutic resistance. This hypothesis is based on published and preliminary data from our group demonstrating that TAMs and TAFs are abundantly present in an inflammatory subtype of NB at diagnosis associated with a high risk of recurrence and extremely poor prognosis. We also have evidence that TAMs and TAFs when exposed to tumor cells stimulate their proliferation, survival and drug-resistance via the paracrine production of pro-tumorigenic cytokines and chemokines that activate in tumor cells signaling pathways such as STAT3 and ERK. Our project has 3 aims. Aim 1, will examine mechanisms of cooperation between TAMs and TAFs, testing the hypothesis that in MYCN amplified tumors that do not produce the TAM chemoattractant CCL-2/MCP-1, TAFs are a source of this chemokine. We will also examine the contribution of cytokines and chemokines generated in co-culture of TAMs, TAFs and NB cells and the signaling pathways they activate in NB cells leading to increased proliferation and survival. Aim 2, will examine changes in the TME landscape secondary to chemotherapy in syngeneic murine NB models (with Project 2) and validate the data in patient tumor samples obtained via Core B. By examining changes in the transcriptome that occur in NB cells chronically exposed to TAM/TAF and their potential epigenetic origin (with Project 3), aim 2 will also identify vulnerabilities to prevent resistance to chemotherapy or targeted therapy (with Project 1). Aim 3, will then translate these discoveries in pre-clinical tumor models. We will test the therapeutic efficacy of the most promising agents targeting TAFs, TAMs, or pathways they activate in tumor cells in combination with chemotherapy or immunotherapy (with Project 5), using human NB lines and patient-derived xenotransplants in immunodeficient mice as well as murine cell lines in immunocompetent mice (with Project 2), The most effective agent(s) will then be proposed for early phase clinical trials to the NANT (Core B). Thus Project 4 brings a unique contribution to the overall objective of this PPG through its focus on the TME and on non-autonomous mechanisms leading towards therapeutic resistance and immune escape.